Process for preparing proteinases



June 25, 1968 Proteinase Activity (0.0 at 670 my) Proteinase Activity (0.0 at 670 mp) KAZUYUKI MORIHARA 3,390,054

PROCESS FOR PREPARING PROTEINASES Filed Dec. 6, 1965 FIG. I

Liquids used for the eiution NoCl NaCl NOCI NaCl NaCl III Fraction Number BO IO ml FIG.2

Hp Proteinase IIe Elastose lilc Crystalline proteinase Concentration of Enzyme: Fr.I.11I 0.005%. Fr.Hp

0.002%. Fr.1Ie 0.2 mg/3 ml INVENTOR W W K m/iwudz United States Patent Oflice 339M Patented June 25, 1968 is to provide a process for pre aring roteinases b the 3 390 054 If t f t t P d p y cu iva ion s rains o seu omonas aeruginosa on a PROCESS FOR PREPARING PROTEINASES medium Com prising crude petroleum 011 or a fractlon Kazuyuk ggfigzfigigg fi'g ggfia' ig ga asslgnor to thereof as carbon source. Still another object of the in- Fned e 6 a NOI 2 5 vention is to provide a process for preparing proteinases Claims priority apllhcafiim Japan f; 16, 1964, by the cultivation of strains of Pseudomonas aeruginosa 39/71,200; Mar. 20, 1965, 40/ 16,391 on a medium comprising polyhydric alcohols which are 7 Claims. (Cl. 195-66) secondary products of petroleum gas production as carbon source. A further object of the present invention is 10 to provide a process for preparing proteinase by the cultivation of strains of Pseudomonas aeruginosa on a me- ABSTRACT OF THE DISCLOSURE dium which additionally comprises yeast extract as its nitrogen source. A still further object of the invention is Proteinase is prepared by inoculating a nutrient medito provide a process for preparing proteinase which has um which contains, as sole carbon source, a non-carbo an elastolytic activity besides its proteolytic activity. hydrate (e.g. heavy mineral oil or aliphatic polyhydric Other objects and attendant advantages of the present alcohol), with a strain of Pseudomonas aeruginosa, culinvention will be apparent to those skilled in the art from turing and recovering accumulated proteinase from the the following disclosure and the appended claims. culture broth. For the carbon sources employed for the process of the present invention, various kinds of hydrocarbons and polyhydric alcohols can be utilized depending on the nature of the strain and the cultivation conditions em- The P3 inventlon relate? to a Proces? for P ployed. For the purpose of illustration, the following delng Protemases, and more partlculafly the lflventlon tailed description is based on typical culture broth comlales a I10V@1 Process for P P F by positions and a strain of Pseudomonas aeruginosa which oculating a strain of Pseudomonas aeruginosa in a medihas been Supplied by the Institute for Fermentation, um which comprises non-carbohydrate carbon sources Osaka and which bears accession number 2 3455 together with suitable nitrogen sources and inorganic TWO kinds of culture broth have been illustratively Salts, and recovering accutnulated, mots/{Haw from the employed;mediumAhas been used to illustrate the growth Cultured broth after mcfilbatlon Penodvs. the variance of the carbon source that is derived from There has heretofore Pnmanly been employed hlghly petroleum resources and medium B has been used to ilconcentfated solution of carbohydrate for example a lustrate the growth vs. that of the carbon source that is glucose solution of high concentration, for high potenderived from petroleum gas production as a Secondary tial production of proteinase by cultivation of a strain of product glycerol being appended for the purpose of Pseudomonas aeruginosa on a medium comprising nonparisom believed that the incorporation of some kind of carbohydrate in the culture medium is indispensable. This has MEDIUM A rendered the prior processes unduly expensive, since carbohydrates are inherently expensive and limited in view carbim soflrce denved from Vanous concentrations of their nature as natural resource. 40 g g h dro en hos hate gg The disadvantage of the prior procedure is obviated by Potassium g plgwspfiate p the present invention, according to which proteinase can Magnesium Sulfate (7H 0) 1 be produced and accumulated favorably in a culture Calcium carbonate 2 20 2-07 broth comprising a component derived from petroleum Tween 203 6 resources as sole carbon source, by the culturing in such Tap Water Balat'lce broth of an inoculated strain of Pseudomonas aeruginosa. "'T Furthermore, according to this invention, aliphatic polyz f if fggg if ggg blg a f g g i fiw g gg for hydric alcohols which are available as secondary products the heavy oil concentrations of 1%, 3%, 7% and 10%. of petroleum gas production can be utilized in culture Surfactantmedia as suitable carbon sources and the proteinases ob- IUM B tained with such media show approximately twice as gigg ggggg acnvlty as those Obtamed by utlhzmg Carbon source derived from Various concentrations, The present invention thus, briefly stated, is a process Fr jfgf gg i nitrate g for preparing proteinase which comprises inoculating Potassium 'i Pseuzlomonas aeruginosa on a medium .comprising non- Potassium diba hosph e saccharide carbon sources, accumulating proteinase in Magnesium 2 iiicclmrlrrirrledium and extracting said protemase from said Manganese chloride (4H2O) n 004 The primary object of the present invention is to progggf s i g vide a process for preparing proteinases by the cultiva- Distilled water Balance tion of strains of Pseudomonas aeruginosa on a medium r t rbo trient as i Norm-Calcium carbonate is added in amount as much as compnsmg non carbohyd ca n nu e 0.3%, 0.5%, 0.7% and 1.0% respectively fOl the respective carbon source. Another ob ect of the present invention glycpm.conoentl-uflons of 1%, 3%, 5% and 3 Using shaking flask of 500 ml. capacity, the afore-mentioned strain is inoculated on 10 ml. of the medium A and B with various concentration of carbon source and submerged for -10 days at 27 C. to obtain the following results. 0

TAB LE I Carbon sources Concentration Growth [PU] 11.

(percent by weight) X- Heav etroleum oil. 1 0. 3

y p 3 n. s 10 7 1. 5 10 1. G 7 O. 5 7 0. O 1 :l: 0. l 3 (l. O 1 0. 3 3 l. 0 n-Propanol 1 i 0. 1 Isopropanol. 1 O. 0 N-butanol 1 0.0 1 :l: 0. 2 Ethyleneglycol 3 0. 0

7 0. 0 2 10 0. 0 1 O. 6 lropyleneglyeol 3 O. 8 7 0. 0 10 O. 0 i, ii+ Glycerol N oTE.Prote0lytic activity was determined using casein as substrate by the method described previously (K. Morihara, J. Bacteriol., 88, 745 (1964), in which the specific activity ([PU1) is expressed as milligrams of tyrosine released per minute.

As is shown by the above table, the maximum proteinase production obtained by the strain with the medium containing petroleum carbon source is at 710% concentration and results in approximately and the maximum production obtained with polyhydric alcohols is glycerol 3%5% concentration which results in 2.8x l0- [PU] /ml.

In the case of heavy petroleum oil, the amount of proteinase production and the concentration of the hydrocarbon in the broth are in a linear relationship whereas in case of polyhydric alcohols there is no such relationship. In addition, the growth of bacterium cell and the rise in the proteinase unit of the product are not necessarily in a proportional relationship.

From the standpoint of economical and industrial usefulness, it is rather practical to utilize heavy petroleum oil and propyleneglycol in spite of the fact that the latter shows a rather poor result in the above table. However, this can be compensated for by the addition of yeast extract as nitrogen nutrient, as shown in the following table:

The general procedure of preparing proteinases and the ph-ysico-chemical properties of the end products of the present invention will be summarized in the following.

For the starting materials of the present invention, high boiling point fractions of petroleum hydrocarbons and polyhydric alcohols of petroleum origin are the most preferable, although other hydrocarbons and polyhydric alcohols can be utilized.

Although yeast extract has been particularly dealt with as nitrogen source and nitrogen containing nutrient, various other ammonium salts of organic and inorganic origin, that is, for instance, ammonuim sulphates, peptone, meat extracts, yeast extract, corn steep liquor, hydrolyzates of casein and the like, can also conveniently be used. Further, suitable phosphates, salts of magnesium, calcium and strontium can be incorporated in the broth.

These materials can be suitably selected and combined for the medium depending on the nature of the strains employed and the conditions used for the incubation of the strain.

A liquid type medium will be advantageously employed in a stationary culture and tank submersion with or without agitation and/ or bubbling. However, tank submersion is especially preferable for mass treatment.

The cultivation will preferably be carried out at 30 40 C. in an aerated or agitated condition. The submersion period can be shortened with rise in the culture broth temperature; thus, for instance, an optimum result, Le. a maximum enzymatic factor will either be attained in a submerging period of five days at 30 C. or in a period of fewer (2-3) days at 37-40 C.

For the extraction of proteinase from the culture broth, any per se conventional refining means can be employed; that is, the proteinase can be obtained in crystalline form by operations such as salting out with ammonium sulphate, acrinol precipitation, fractional sedimentation as Well as column chromatography with cation exchange resin or DEAE-cellulose etc.

For example, three species of proteinase as illustrated in FIG. 1 can be isolated by per se known fractional column chromatography utilizing DEAE cellulose from centrifuged supernatant salted out with ammonium sulr phate and containing proteinases which have been formed by five days submersion of Pseudomonas aeruginora in a culture medium containing hydrocarbons originated from petroleum or polyhydric alcohols originated from petroleum gas, as carbon source.

After dial-yzing against water followed by freeze drying, the various resultant proteinase fractions 1, II and III are obtained.

FIG. 2 shows the observed effect of pH values for enzymatic activity, in which the optimal pH values for the fractions 1, II and III are found as 6.5, 7.8 and 9.5; therefore, they are regarded as neutral, weak basic and basic proteinase respectively. The data given in Table 3 express the protein decomposing ability of these three proteinases versus the properties of the substrates employed and prove the differences in specificities of these three proteinases. Moreover, fraction II has elastolytic activity besides proteolytic activity. Fraction III has a specificity which is quite similar to that of known crystalline proteinase, and a further study of the physicochemical properties of the crystalline enzyme establishes that fraction III is entirely the same as the known proteinase described in British patent specification No. 967,- 848.

TABLE 3.ENZYMATIC ACTIVITIES OF THESE P ROTE INASES Enzyme Fr. Proteinase Elastaso Gel]. [PUlGus 2 ma 3 Sachars Method (L. A. Saehar et al. Proc. Soc. Exp. Biol. Med. 90,

323 (1955)) with oreein elastiu 20 mg in 0.1 mol Tris buffer (pH 7.5) lml.

for enzyme solution 1 n1l. and water 1 ml., shaked for 3 hours at 40 C. [E U Elastase Unit, 1S defined as milligrams of dissolved elastin per milligrams of enzyme used.

6 Example 1 Pseudomonas aeruginosa (IFO No. 3455 strain) is inoculated on an aqueous medium comprising of TABLE 4.-STABILITIES OF THESE THREE PROTEINASES [Measured as percent of retained activity] pH values of environment 1 Temperature, C. 2

Fr. Enzymatic Activity I Proteinase. 0 90 100 100 100 88 0 100 96 63 0 II Proteinase. O 0 100 100 76 0 100 90 89 80 41 0 Elastase 0 0 33 100 100 76 0 100 90 86 71 0 III Proteinase. 0 87 100 100 100 93 0 100 94 85 0 1 Against pH values oi environment (Concentration of enzyme: 0.01% after being allowed to stand overnight at room temperature). 2 Against Temperature (Concentration of enzyme: 0.01% for 10 minutes at pH 7.5).

TABLE 5.EFFECTS 0F CHELATING AGENT AGAINST THESE THREE PROTEINASE [Concentrations of enzyme: 0.01% for 30 minutes at 40 (1.]

Measured as percent of retained activity These deactivated enzymes can be reactivated again by dialysis against water.

Furthermore, these three proteinases will not be affected by inhibitors such as diisopropyl phosphofluoridate (10 mol) hydrogen cyanide (10' mol), thioglycolic acid (10- mol), p-chloromercury benzoate (10- mol), and by these inhibitors extracted from soybean or potato.

The elastolytic activity of fraction II which goes along with its proteolytic activity cannot be separated from the latter by fractionation with DEAE cellulose; further the optimal pH values for the stabilities of and the behaviors to chelating agents of the two activities are identical. Proteinase is recoverable in needle-like crystalline form from fraction II by introducing ammonium sulfate. Since it has been confirmed that fraction II is a single substance, by means of ultra-centrifuge and electrophoresis in spite of the fact that it has an elastolytic activity together with proteolytic activity, it has been concluded that the two activities are ascribed to the same enzyme protein.

The physico-chemical properties of the crystalline enzyme isolated from fractions II and III which are end products of the present invention are summarized in the following table, fraction I being omitted from the table because only qualitative and not reliable quantiative results could be obtained therewith.

TABLE 6.PHYSIOOCHEMICAL PROPERTIES OF THE CRYSTALLINE ENZYMES 3 sparingly soluble. 4 Readily soluble.

Presently preferred embodiments of the invention are set forth as follows, solely for the purpose of illustration. Percentages therein are by weight.

heavy petroleum oil, 1% of diammonium hydrogen phosphate, 1% of disodium hydrogen phosphate, 0.2% of potassium phosphate, 0.1% of magnesium sulfate (7H O), 1% of yeast extract, 0.7% of calcium carbonate and 0.05% of surfactant (Tween 20) and being adjusted to pH 7. Aerated cultivation with agitation is then carried out for five days at 37 C. An activity of 4X10- [PUJ/ml. is obtained. Thus obtained crude broth is salted out by the introduction of saturated aqueous solution of ammonium sulphate. Then the supernatant of said liquor is dialyzed against a buffer solution of 0.02 molar phosphoric acid (pH 8) and is applied. to a DEAE cellulose column which has previously been treated with 2 liters of said buffer. The said column is subsequently eluted with 2 liters of said buffer, 2 liters of 0.1 molar sodium chloride solution (adjusted with 0.02 molar phosphoric acid buffer, pH 7.0) and 2 liters of 0.3 molar sodium chloride solution (adjusted with 0.02 molar phosphoric acid buffer, pH 7.0) to afford the eluates of three different fractions I, II and III in succession. The inclusive yield of this column chromatography is and the elution gives the fractions I, II and III in 6, 68 and 26% yield respectively. After refrigeration needle crystals are formed from the water-diluted eluate of fraction II by concentrating below 40 C. followed by salting out with ammonium sulphate solution to such an extent that the eluate is slightly clouded. Fraction III is recovered from the eluate by the following procedure, that is the eluate containing fraction III is dialyzed against water and to this, 1% acrinol is added so as to make the concentration to 0.07%. The precipitate is collected by centrifugation, rinsed with water and extracted with 0.5 molar calcium chloride solution. The extract is then treated with 0.5 to 2 volumes of acetone in a refrigerator (at a temperature lower than 5 C.) to fractionally precipitate crude enzyme. The precipitated crude enzyme is dissolved in 0.01 molar calcium chloride solution so that the concentration of protein is as high as possible. After removal of insoluble substances, acetone is introduced dropwise into the suspension while stirring, until a slight turbidity appears. After being allowed to stand overnight in a refrigerator, more acetone is added dropwise to the supernatant whereby needle crystals of Fraction III separate.

The physico-chemical properties of these crystalline enzymes are as aforementioned in Tables 3-6.

1 Example 2 Pseudomonas aeruginosa (IFO No. 3455 strain) is inoculated on an aqueous medium comprising 5% of propyleneglycol, 1% of secondary ammonium phosphate, 0.2% of potassium phosphate, 0.05% of magnesium sulphate, 0.5% of calcium carbonate and 1% of yeast extract and being adjusted to pH 7. Aerated cultivation with agitation is then carried out for five days at 37 C. and an enzymatic activity of 16.0 10" PU] /ml. is obtained.

Fractionation is carried out according to the procedure of Example 1.

What is claimed is:

1. A process for preparing proteinase which comprises cultivating Pseudomonas aeruginosa under aerobic conditions in a synthetic culture medium containing, as its sole carbon nutrient, non-carbohydrate selected from the group consisting of heavy mineral oil and aliphatic polyhydric alcohols, and thereafter separating the accumulated proteinase from the nutrient medium.

2. A process as claimed in claim 1, wherein said noncarbohydrate nutrient is heavy mineral oil.

3. A process as claimed in claim 1, wherein said noncarbohydrate carbon nutrient is aliphatic polyhydric alcohol.

4. A process as claimed in claim 2 wherein said synthetic culture medium contains nitrogen nutrient.

References Cited UNITED STATES PATENTS 3,222,258 12/1965 Iizuka et al. 19529 3,234,105 2/1966 Motizuki et al. 195-49 3,271,266 9/1966 Laine et al. 195-3 LIONEL M. SHAPIRO, Primary Examiner. 

